DESCRIPTION: The bifunctional enzyme, 6-phospho-2- kinase/fructose-2,6-bisphosphatase is the sole enzyme responsible for the synthesis and degradations of fructose 2,6-bisphosphate (F2,6-P) a potent modulator of hepatic carbon flux. F2,6-P is an allosteric activator of the glycolytic enzyme 6-phosphofructokinase and an inhibitor of the gluconeogenic enzyme fructose 1,6-bisphosphatase. The metabolic effects of glucagon, via cAMP-dependent protein kinase, on hepatic carbon flux are mediated by the intracellular concentration of F2,6-P. In diabetes, excessive production of glucose by the liver is a major contributor to hyperglycemia, which leads to the major problems associated with the disease. The central role of F2,6-P in control of hepatic glucose production and utilization suggests that drug therapies directed towards increasing F2,6-P levels will be beneficial to the diabetic patient. The proposed studies will investigate the efficacy of targeting the bisphosphatase domain of the bifunctional enzyme for inhibition therefore increasing the levels of F2,6-P using two strategies: 1) production of transgenic mice that express a mutant enzyme or carry a knockout enzyme gene, to establish chronically high or low F2,6-P levels, respectively, and 2) physical studies using NMR spectroscopy to characterize the reaction mechanism of the bisphosphatase domain and define the functional role of active site amino acid residues. This should provide the basis for the rational design of specific inhibitors of the bisphosphatase activity of hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.